Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

Copine proteins are highly conserved and ubiquitously found in eukaryotes, and their indispensable roles in different species were proposed. However, their exact function remains unclear. The phytohormone brassinosteroids (BRs) play vital roles in plant growth, development and environmental responses. A key event in effective BR signaling is the formation of functional BRI1-SERK receptor complex and subsequent transphosphorylation upon ligand binding. Here, we demonstrate that BONZAI (BON) proteins, which are plasma membrane-associated copine proteins, are critical components of BR signaling in both the monocot maize and the dicot Arabidopsis. Biochemical and molecular analyses reveal that BON proteins directly interact with SERK kinases, thereby ensuring effective BRI1-SERK interaction and transphosphorylation. This study advances the knowledge on BR signaling and provides an important target for optimizing valuable agronomic traits, it also opens a way to study steroid hormone signaling and copine proteins of eukaryotes in a broader perspective.

As introduced above, nearly all of the major BR signaling components were originally identified in the model plant Arabidopsis (Arabidopsis thaliana).However, phylogenetic studies indicate that these genes are highly conserved among monocots and dicots 1 , suggesting that the classical BR signaling is conserved at least in monocots and dicots.OsBRI1 28 , OsBAK1 29 , OsGSK1 30 , OsBZR1 31 , ZmBRI1 8 , and ZmBZR1 32 , the orthologs of the known Arabidopsis genes in rice and maize (Zea mays), have been demonstrated to have conserved functions as in Arabidopsis.Other genes conservative in BR signaling among monocots and dicots remain to be identified and characterized.
Copines are a group of highly conserved proteins ubiquitiously found in various eukaryotes including Paramecium teraurelia, plants, Caenorhabditis elegans, mouse, and human [33][34][35][36][37] .The conserved nature of copines in different organisms suggest that they play important roles in common biological pathways, which is supported by emerging studies demonstrating their roles in plant development, defense and stress responses [38][39][40][41][42][43] .All copines are characterized by two C2 domains at their N termini and a von Willebrand A domain at their C termini 33,44 .While the C2 domains are Ca 2+ -dependent phospholipid-binding domains implicated in membrane association, the A domain is thought to be involved in protein-protein interactions 34,[45][46][47] , may thus play a role in protein complex formation and signaling.
Further characterization indicated that the TIR (Toll interleukin 1 receptor)-nucleotide-binding, leucine-rich repeat (NB-LRR) (TNL) type resistance (R) gene SNC1 (SUPPRESSOR OF npr1-1, CONSTITUTIVE1) was present in the Col-0 accession but not in the Ws accession and was responsible for the accession-dependent dwarf and autoimmune phenotypes of bon1-1 mutants 38,39 , and several other TNL genes also contribute to the autoimmunity triggered by loss of BON1 and BON3 functions 48 .Moreover, TNL-type R gene-mediated disease resistance was shown to be dependent on PHYTOALEXIN DEFICIENT4 (PAD4), which encodes a lipase-like protein 49 , with the pad4-1 mutation fully suppressing the dwarfism of bon1-1, and bon1-1 bon3-3 38,39 .However, the bon1-1 bon2-2 bon3-3 pad4-1 quadruple mutant still exhibited obvious growth defects even though the autoimmunity was fully suppressed 39 .Therefore, BONs' function in plant immunity might be less conserved, and their conserved intrinsic function independent of SNC1/TNLs-and PAD4-mediated autoimmunity needs to be uncovered.For years, the attempt to identify such function has been impeded by gene redundancy of BON members and the existence of those TNL-type R genes in Arabidopsis.
Here, by studying the BON homologous genes in maize, we discovered that the loss of ZmBON1 function led to a dwarf morphology not caused by autoimmunity but by deficient BR response.Consistently, the Arabidopsis bon1-1 bon2-2 bon3-3 pad4-1 mutant showed compromised sensitivity to BR treatment, indicating that BON proteins function in BR signaling.Further study indicates that BON proteins are required for BRI1-SERKs protein complex formation and the subsequent transphosphorylation of BR signaling components, thus acting as critical regulatory components of BR signaling in both monocot and dicot plants.

Loss of ZmBON1 function leads to a dwarf phenotype in the monocot maize
Since the existence of TNL-type R proteins impedes the functional analysis of BON functioning in the dicot Arabidopsis, we sought to characterize the function of BON proteins in the monocot maize, wherein no TNL-type R genes exist.To this end, we screened the maize genome, and only two genes ZmBON1 (GRMZM2G494514) and ZmBON3 (GRMZM2G176995), which show close homology to BON1 and BON3 in Arabidopsis, were identified (Supplementary Fig. 1).We utilized clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing to generate Zmbon1 and Zmbon3 single mutants and Zmbon1 Zmbon3 double mutants by targeting ZmBON1 and ZmBON3 alone or simultaneously in the maize inbred line KN5585.We obtained more than six different allelic mutant lines for each construct, with insertions or deletions of 1 to 16 bp generated near the protospacer adjacent motif (PAM) but no mutation in potential off-target sites (Supplementary Fig. 2).All Zmbon1 mutant lines showed drastic dwarf morphology, while none of the Zmbon3 single mutants showed obvious differences from wild-type plants (Fig. 1a, b).The Zmbon1 Zmbon3 double mutants exhibited enhanced dwarfism compared to the Zmbon1 mutants, indicating that ZmBON3 functions additively with ZmBON1.All phenotypes were heritable after seven generations of planting in the field (Supplementary Fig. 3).
To determine whether the observed phenotype is consistent in different genetic backgrounds, we identified two EMS mutants in the B73 background for ZmBON1 and ZmBON3, namely Zmbon1-7 and Zmbon3-7, respectively, wherein point mutations were predicted to result in truncated proteins (Supplementary Fig. 4a, b).Like the Zmbon1 and Zmbon3 mutants in the KN5585 background, Zmbon1-7 showed obvious dwarfism, while Zmbon3-7 showed no morphological differences from the wild-type B73 (Supplementary Fig. 4c).
We then carried out transcriptome deep sequencing (RNA-seq) of the 5-week-old wild-type KN5585 and Zmbon1-1 plants.Defense-related genes were not significantly enriched among genes upregulated in Zmbon1-1 relative to KN5585 (Supplementary Fig. 5, Supplementary Data 1).We also analyzed the mutant for expression of the immune marker genes ZmPR1 and ZmPR5, which had expression changes in the range of 10-100 times in the maize autoimmune mutants Zmgb1 CR (a mutant in a heterotrimeric G protein β subunit gene) and lls1/les30 (lethal leaf spot 1/lesion mimic 30) [50][51][52] .We observed no significant differences in ZmPR1 or ZmPR5 gene expression in the Zmbon1-1, Zmbon3-1, or Zmbon1 Zmbon3-1 mutant compared to KN5585 at different growth stages, when the Zmbon1-1 mutant constantly showed obvious dwarf phenotype (Fig. 1e, Supplementary Fig. 6).Moreover, the level of the defense phytohormone salicylic acid (SA) did not change in the Zmbon1-1, Zmbon3-1, or Zmbon1 Zmbon3-1 mutants (Fig. 1f).The above data indicate that the dwarf phenotype is not associated with autoimmune responses as in Arabidopsis.
ZmBON1 is involved in BR signaling in maize Morphological analysis showed that the dwarfism of Zmbon1-1 was caused by shorter rather than fewer internodes (Supplementary Fig. 7a).In addition, the leaves of Zmbon1-1 were disordered and twisted.The leaf angle of Zmbon1-1 had a larger variation among different leaves compared to that of the wild type (Fig. 2a), and somefield-grown plants showed an obvious corkscrew appearance (Supplementary Fig. 7b).Moreover, the hundred-kernel weight was significantly reduced in Zmbon1-1, suggesting that the mutant produces smaller kernels (Fig. 2b).All these phenotypes are typical characteristics of maize BR-defective mutants, such as the ZmBRI1-RNAi lines 7,8,[53][54][55] .We thus suspected that the growth defects of Zmbon1 might be related to deficiency in BR signaling.
To test this hypothesis, we performed a root growth inhibition assay.Upon treatment of 100 nM 2,4-epibrassinolide (eBL), one of the most potent BRs, the root growth of KN5585 was largely inhibited.By contrast, the inhibition of root growth was significantly reduced in the Zmbon1-1 mutant (Fig. 2c, d), indicating that BR sensitivity in Zmbon1-1 is lower.Consistently, the Zmbon1-1 mutant in the B73 background also showed reduced eBL sensitivity (Supplementary Fig. 8).The maize BR biosynthetic marker genes, Brassinosteroid dependent1 (BRD1) and Constitutive photomorphogenic dwarf (CPD) 8 , were significantly upregulated in Zmbon1-1 as seen by reverse transcription-quantitative PCR (RT-qPCR) (Fig. 2e), another hallmark of BR-insensitive mutants 8 .These data indicate that ZmBON1 is involved in BR signaling in maize.

BON's involvement in BR signaling is conserved in the dicot Arabidopsis
We tested whether the function of BONs in BR signaling is conserved between the monocot maize and the dicot Arabidopsis by conducting a root inhibition assay with the Arabidopsis mutant bon1-1.Exogenous application of 2 nM, 5 nM, or 10 nM eBL all significantly inhibited root growth in the wild-type Col-0, Ws, and the pad4-1 mutant at 12 days after treatment (Supplementary Fig. 9b, c and Fig. 3b, c).The 10 nM eBL significantly inhibited root growth in both the Col-0 and bon1-1 seedlings to a similar extent (Supplementary Fig. 9b, c).The 10-nM eBL application also equally inhibited the root growth of wild-type Ws and bon1-2 seedlings (Supplementary Fig. 9b, c).Moreover, the bon1-2 bon3-1 (Ws) double mutant also showed the same level of root inhibition as its wild-type Ws upon eBL treatment (Supplementary Fig. 9b, c).We further analyzed the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 mutants, as the autoimmunity-conferred lethality phenotype of the bon1-1 bon2-2 bon3-3 triple mutant is largely suppressed by the pad4-1 mutation (Fig. 3a, Supplementary Fig. 9a).There was no difference in the degree of inhibition between pad4-1 and Col-0 seedlings upon eBL treatment, indicating that PAD4 is not involved in BR signaling (Supplementary Fig. 9b, c).Intriguingly, eBL treatment caused a dramatically weaker root inhibition in the bon1-1 bon2-2 bon3-3 pad4-1 mutant than in the pad4-1 mutant when the phytohormone was provided as a 2-nM (Fig. 3b, c), 5-nM and 10-nM (Supplementary Fig. 9b, c and Fig. 3c) concentration, implying a defective BR signaling in the bon1-1 bon2-2 bon3-3 pad4-1 mutant.In addition, the bon1-1 bon2-2 bon3-3 pad4-1 mutant displayed significantly shorter hypocotyls than the pad4-1 mutant when grown in the dark (Fig. 3d, e), further supporting the notion of the impaired BR signaling when BON genes were fully knocked out in Arabidopsis, since hypocotyl elongation in the dark is dependent on active BR signaling 5 .

BONs interact with SERKs in Arabidopsis and maize
To elucidate how BON proteins regulate BR signaling, we performed a yeast two-hybrid (Y2H) screen for BON1-interacting proteins.Since the Arabidopsis BON1 is a plasma membrane-localized protein 34 , and the C-terminal A domain of BON1 (BON1-A, from Val-319 to Pro-578) is responsible for protein-protein interactions 34,46,47 , we used it as a bait to screen an Arabidopsis cDNA library.We identified SERK4, which was shown to work redundantly with SERK3 in BR signaling 19 , as a candidate interactor; we confirmed this interaction by retransformation in the yeast two-hybrid assay (Fig. 5a).We also independently validated the interaction between BON1 and SERK4 by a coimmunoprecipitation (Co-IP) assay in Arabidopsis protoplasts transiently transfected with the BON1-HA and SERK4-FLAG constructs (Fig. 5b) and by a split luciferase complementation (SLC) assay using a Nicotiana benthamiana transient expression system (Fig. 5c).Because BON1 was also previously shown to interact with SERK3 46 , we tested two other members of the SERK family, SERK1 and SERK2 19 , for their interaction potential with BON1.Both Y2H and SLC assays indicated that SERK1 and SERK2 also interact with BON1 (Fig. 5a, Supplementary Fig. 11).BON1 did not appear to interact with other signaling components, including BRI1, BKI1, BIK1, TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE1 (TTL1), TTL4, BSK1, and BSK3, in the Y2H (Supplementary Fig. 12).Similar to the Arabidopsis proteins, ZmBON1  interacted with ZmSERK4 (encoded by GRMZM2G384439), a close homolog of SERK4 in maize, in the Y2H (Fig. 5d) and SLC assays (Fig. 5e), indicating that the interaction of BON1 and SERKs is conserved in Arabidopsis and maize.Interestingly, although BON1 did not appear to interact with BRI1 in Y2H assay, they associate with each other in the SLC assay (Fig. 5f) and Co-IP assay (Fig. 5g).These data suggest that BON1 and BRI1 are physically close to each other although they may not interact directly.

BON proteins are critical for the formation of the BRI1-SERK complex
Since BON proteins directly interact with SERKs and indirectly interact with BRI1, we wonder if BON proteins may affect the stability of BRI1 and SERKs.The abundance of BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 were detected at 0, 30, and 60 min after eBL treatment using anti-BRI1 and anti-SERK3 antibodies (Supplementary Fig. 13a, b), the immunoblot data indicated that the abundance of BRI1 and SERK3 were not altered in bon1-1 bon2-2 bon3-3 pad4-1 compared to that of pad4-1 (Supplementary Fig. 14).
As the endocytosis of SERKs play important role in BR signaling 57 , we then carefully examined the subcellular localization of BON1.Interestingly, other than the typical plasma membrane-localization pattern observed in previous studies 34,45 , the granule signal of BON1 associated with the plasma membrane or in the cytosols was observed by switching the focal plane of the confocal (Supplementary Fig. 15a).Furthermore, the SERK4 exhibited similar localization pattern with BON1 (Supplementary Fig. 15b).Since BON1 directly interacts with SERKs, we asked whether BONs affect the trafficking of SERK proteins.However, when comparing the localization pattern of SERK3 and SERK4 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 we didn't find significant differences (Supplementary Fig. 15c, d).To further confirm BONs' effect on subcellular localization of SERK proteins, we purified the total plasma membrane proteins of pad4-1, bon1-1 bon2-2 bon3-3 pad4-1, SERK4-FLAG/pad4-1, and SERK4-FLAG/ bon1-1 bon2-2 bon3-3 Fig. 5 | BONs interact with SERKs and BRI1.a Yeast two-hybrid (Y2H) assay assessing the interaction of the A domain of BON1 with the kinase domain (KD) of SERK4, SERK1 and SERK2.The constructs were co-transformed into yeast cells and grown on selective dropout medium as indicated.b Co-IP assay of BON1-HA and SERK4-FLAG in Arabidopsis protoplasts transfected with the respective encoding constructs.BON1-HA and SERK4-FLAG proteins were detected using anti-HA and anti-FLAG antibodies, respectively.PIN1-HA was used as a negative control.c Split luciferase complementation (SLC) assay showing the interaction between BON1 and SERK4.PIN1 was used as a negative control.d Y2H assays showing the interaction between ZmBON1 and the ZmSERK4-KD.e SLC assay showing the interaction between ZmBON1 and ZmSERK4 in N. benthamiana leaves.ZmCDPK7 was used as a negative control.f SLC assay showing the interaction between BON1 and BRI1 in N. benthamiana leaves.PIN1 was used as a negative control.g Co-IP assay of BON1-HA and BRI1-GFP in Arabidopsis protoplasts transfected with the respective encoding constructs.BON1-HA and BRI1-GFP proteins were detected using anti-HA and anti-GFP antibodies, respectively.PIN1-HA was used as a negative control.All experiments were repeated 3 times with similar results.
pad4-1, and compared the abundance of SERK3 and SERK4-FLAG by immunoblots.Again, no significant difference of SERK3 or SERK4-FLAG protein level was observed (Supplementary Fig. 15e, f).
To confirm the data from the above quantitative phosphoproteomics analysis, we isolated total proteins from mock-and eBLtreated pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 plants, and immunoprecipitated the BRI1 and the SERK proteins using their related antibodies, followed by immunoblots using the anti-phosphoserine (anti-pSer) and anti-phosphothreonine (anti-pThr) antibodies to detect the general phosphorylation changes of BRI1 and SERKs.As confirmed by three biological replicates, the general level of phosphorylated BRI1 proteins increased in pad4-1 seedlings in response to eBL, while eBL-induced BRI1 phosphorylation was largely compromised in bon1-1 bon2-2 bon3-3 pad4-1 seedlings relative to pad4-1 (Fig. 6f, g and Supplementary Fig. 17a, b).We also confirmed the phosphorylation change of BRI1 in pad4-1 vs bon1-1 bon2-2 bon3-3 pad4-1 seedlings by detection of proteins extracted from protoplasts transiently expressing BRI1-GFP with four biological replicates (Supplementary Fig. 18).In addition, we co-immunoprecipitated SERK3 and SERK4-FLAG with anti-SERK3 and anti-FLAG antibodies, respectively, and detected the phosphorylated proteins using anti-pThr antibodies.The phosphorylation levels of SERK3 and SERK4-FLAG were also dramatically decreased in bon1-1 bon2-2 bon3-3 pad4-1 seedlings compared to pad4-1 seedlings upon eBL treatment, as confirmed by three biological replicates (Fig. 6h, i and Supplementary Fig. 17c, d), which was consistent with the results of the quantitative phosphoproteomics.The above data support the idea that BON proteins are critical regulators of the reciprocal phosphorylation between BRI1 and SERKs.

Discussion
In this study, by characterization of the maize Zmbon1 mutant wherein no autoimmunity is triggered since no TNL-type R genes exist in maize, we found ZmBON1 was involved in BR signaling (Figs. 1 and 2).Moreover, the conserved function of BONs in BR signaling was validated using Arabidopsis bon1-1 bon2-2 bon3-3 pad4-1 quadruple mutant wherein the pad4-1 mutation blocked the autoimmunity and rescued the lethal phenotype of bon1-1 bon2-2 bon3-3 (Figs. 3 and 4).While the autoimmunity of bon mutants in Arabidopsis is caused by the activation of TNL-type R proteins, the growth defect in maize and Arabidopsis is largely due to the deficient BR signaling.Our studies in both monocot and dicot plant systems thus uncovered the intrinsic function of BONs in BR signaling beyond their less conserved role in plant immunity.Since BR is also involved in growth-defense coordination 58 , it will be worthy of investigation if and how BON proteins could mediate the interplay between BR signaling and plant immunity.
Our data indicate that BON proteins are critical regulatory component for proper functioning of BR receptor complex (Fig. 7).Transcription of BR-related genes including CPD, ROT3, BR6OX2, DWF4, BAS1, and SAUR-AC1, etc. were significantly altered in bon1-1 bon2-2 bon3-3 pad4-1 and bak1-4 compared to pad4-1 and Col-0 wild type upon eBL treatment (Fig. 4, Supplementary Fig. 10), these genes were also identified in previous transcriptome analysis of BR responses [59][60][61] , implying the important role of BON proteins in BR signaling.In addition, BON proteins interacted directly with the BR co-receptor SERKs and indirectly with BRI1, and thus likely form a BON-SERK-BRI1 complex (Fig. 5).BON proteins do not appear to affect the protein stability and trafficking of BR receptor complex (Supplementary Fig. 14 and Supplementary Fig. 15).Instead, BON proteins likely act as a chaperone or structural proteins promoting the interaction between BRI1 and SERKs upon ligand binding (Fig. 6).When BON proteins were depleted, the BR-triggered interaction between BRI1 and SERKs was largely attenuated (Fig. 6).In support of the above notion, BR-triggered transphosphorylation of BRI1 and SERK proteins were largely reduced in the bon1-1 bon2-2 bon3-3 pad4-1 mutant, as detected by both phosphoproteomics analysis and immunoblotting (Fig. 6, Supplementary Figs.14, 16, 17, 18).The phosphorylation of BKI1, BSKs, and BSLs and dephosphorylation of BZR1 were also attenuated in the bon1-1 bon2-2 bon3-3 pad4-1 mutant (Supplementary Fig. 16, Fig. 3).Therefore, BON proteins are involved in the regulation of entire BR signaling pathway via modulating the functioning of BR receptor complex.According to previous studies, BON1 was known to be associated with the detergent-resistant microdomains and its localization in plasma membrane is essential for BON1's function 45 .Interestingly, recent studies have found that the existence of preassembled BRI1-SERKs complexes in the microdomains of plasma membrane 62,63 .Further research is needed to investigate if BON proteins act as a structural protein affecting the microdomain formation or attracting the SERKs proteins to transport to the microdomain of plasma membrane, thereby affecting the formation of BRI1-SERKs complex.Besides, we could not exclude the possibility that dephosphorylation and/or autophosphorylation of BRI1 or SERKs are regulated by BON proteins based on current data.
Since copine proteins were ubiquitiously found in different eukaryotes and BR is a steroid hormone that is shared between plants and animals, it is possible copine proteins could act as an evolutionary hub of steroid hormone signaling in different eukaryotes.This hypothesis is at least partly supported by the current finding that BONs' function in BR signaling is conserved in both monocot and dicot.This study thus opens a way for scientists from a broad life science community to explore the potentially similar function of copines in other organisms.In addition, previous structural model of BR receptor complex is based solely on the BRI1, BAK1, and BKI1 [64][65][66][67] .Further structural studies on BON-SERK-BRI1 complex wait to be performed to gain deeper insights into the sophisticated functioning of BR receptor.Moreover, the pleiotropic regulatory effects of BONs render it an important target for optimizing valuable agronomic traits in crops.

Plant materials and growth conditions
For phenotypic observations, maize (Zea mays, inbred lines KN5585 and B73) plants were planted in the field in Zhengzhou (113°66′ E, 34°79′ N) in summer or Sanya (109°17′ E, 18°35′ N) in winter from 2019 to 2022.For laboratory experiments, maize plants were grown at 22 °C under long-day conditions (14-h day/10-h night) in a growth chamber.Arabidopsis (Arabidopsis thaliana) plants from the Col-0 and Ws accessions used in this study were grown at 22 °C under long-day conditions (14-h day/10-h night) in chambers either on potting soil or in sterile Petri dishes containing half-strength solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose and 0.7% (w/v) agar.N. benthamiana plants were grown at 22 °C under long-day conditions (14-h day/10-h night) in chambers, and 3-4-week-old plants were used for transient expression experiments.

Generation of mutants and transgenic lines in maize and Arabidopsis
To generate knock-out mutants of ZmBON1 and ZmBON3, two single guide RNA sequences (sgRNAs) each targeting ZmBON1 or ZmBON3 were cloned into the pBUE411 vector 68 to generate the CRISPR/Cas9 constructs targeting ZmBON1 and ZmBON3 separately or simultaneously.The resulting constructs were introduced into the maize inbred line KN5585 via Agrobacterium (Agrobacterium tumefaciens)-mediated transformation.More than 20 independent lines were generated for each transformation.PCR products covering the target site in the respective genes were sequenced to determine the genotype of all plants.To generate SERK4-FLAG/pad4-1 and SERK4-FLAG/ bon1-1 bon2-2 bon3-3 pad4-1 transgenic plants, the full-length SERK4 coding sequence was cloned into the pSuper1300-35S-3xFLAG-mCherry vector.The resulting construct was transformed into pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 mutant plants using the floral dip method 69 .

Quantification of total salicylic acid (SA)
Fresh above-ground maize tissues from 5-8 seedlings (4-leaf stage) were collected for each sample.Total SA including free SA and SA betaglucoside (SAG) were extracted and detected by MetWare (http:// www.metware.cn/)based on the AB Sciex QTRAP 6500 LC-MS/MS platform following a previously published method 70 .

BR root inhibition assay in maize and Arabidopsis
Maize seeds were soaked on sterilized wet paper towels for 2-3 days until germination.Uniformly germinated seeds were selected and transferred to layered filter paper placed vertically in a pot containing 5.0 L of deionized water alone or containing 20 nM or 100 nM of 2,4epibrassinolide (2,4-eBL; Solarbio, Cat#SB8360).Seedlings were grown at 22 °C under a 14-h day/10-h night photoperiod for 12 days, after which primary root length was measured.Arabidopsis seeds were sterilized with 70% (v/v) ethanol and germinated vertically on halfstrength solid MS medium alone or containing 2, 5, or 10 nM 2,4-eBL at 22 °C for 10 days under long-day conditions before measuring primary root length.The concentration of eBL was chosen according to previous report 4,8 .

RNA-seq analysis
For Arabidopsis, three biological replicates of 3-week-old Arabidopsis seedlings from the Col-0, bak1-4, pad4-1, and bon1-1 bon2-2 bon3-3 pad4-1 genotypes, were mock-treated (0.02% [v/v] Tween-20) or treated with eBL containing 0.02% (v/v) Tween-20 for 30 min.For better absorption of eBL in a short time after spray inoculation, we used a higher eBL concentration (2 μM) in this study.For maize, three biological replicates of leaves from five 5-week-old plants grown in the field of Sanya were collected.All harvested plant tissues were frozen and ground in liquid nitrogen.Total RNA was isolated with RNAiso Plus (Takara, Cat#9109) and used for sequencing on an Illumina NovaSeq 6000 platform.High-quality clean data were mapped to the Arabidopsis reference genome (TAIR10).Gene expression levels were calculated as fragments per kilobase of transcript per million fragments mapped (FPKMs).Differentially expressed genes between two samples were identified using the R package DESeq (1.10.1) based on an adjusted false discovery rate (FDR) < 0.01, and a minimal absolute foldchange ≥1.5.Venn diagram and heatmap were generated using TBtools-II 71 .Statistical enrichment of DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed using on the BMKCloud platform (https://www.biocloud.net) 72.

Phosphoproteomics analysis
Four-week-old pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 Arabidopsis mutant plants were mock-treated (0.02% [v/v] Tween-20) or treated with 2 μM eBL containing 0.02% (v/v) Tween for 30 min.The aboveground tissues were collected, and the liquids on the surface were removed using tissue paper, then tissues of 8 individual plants were grouped as one sample and ground in liquid nitrogen.Total proteins were extracted using SDT buffer (4% [w/v] SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT) and quantified using a BCA protein assay kit (Bio-Rad, USA).Proteins were digested by trypsin according to filter-aided sample preparation (FASP) procedure described previously 73 .Phosphopeptides were enriched using a High-Select TM Fe-NTA Phosphopepetides Enrichment Kit (Thermo Scientific, A32992), concentrated in a vacuum and dissolved in 40 µl of 0.1% (v/v) formic acid solution for mass spectrometry analysis using a timsTOF Pro mass spectrometer (Bruker) coupled to a Nanoelute (Bruker Daltonics).The data were analyzed and quantified using MaxQuant software, and the related parameters and instructions were showed in Supplementary Table 2 (APPLIED PROTEIN TECHNOLOGY).
Yeast two-hybrid (Y2H) screening and protein interaction assays Y2H screening was performed using a Matchmaker Gold Yeast twohybrid system (Clontech).The truncated coding region (BON1-A, encoding the interacting domain) was introduced into the pGBKT7 (BD) vector and used as bait to screen a cDNA library generated from mixed samples of Arabidopsis seedlings infected or not with Pseudomonas syringae pv.tomato DC3000 (Pst).The coding sequence fragments encoding SERK4-KD, SERK1-KD and SERK2-KD and several additional Arabidopsis proteins including BRI1, BKI1, BIK1, TTL1, TLL4, BSK1, BSK3, and BIN2, were individually cloned into the pGADT7 (AD) vector.The gene fragments encoding ZmBON1 and the predicted ZmSERK4/BKK1 (GRMZM2G384439) kinase domain were individually cloned into the pGBKT7 and pGADT7 vector.The constructs were cotransformed in pairs into yeast strain Y2H Gold.The transformants were grown on synthetic complete (SC) medium -Leu/-Trp (Coolaber, Cat#CG5641) and SC medium -Ade/-His/-Leu/-Trp (Coolaber, Cat#PM2113) supplemented with 20 g/L glucose and X-a-Gal to assess interactions between various clone combinations.

Split luciferase complementation (SLC) assays
The coding sequences of BON1 and ZmBON1 were inserted into the pCAMBIA1300-cLuc vector, and the coding sequences of SERK4, SERK1, SERK2, BRI1, PIN1, ZmSERK4, and ZmCDPK7 were inserted into the pCAMBIA1300-nLuc vector.The resulting constructs were transformed into Agrobacterium (strain GV3101).After the bacteria were cultured in liquid LB medium to an OD at 600 nm of 1.2-1.8 at 28 °C, the cultures were pelleted by centrifugation and resuspended in infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl 2 , 150 µM acetosyringone) to a final OD at 600 nm of 1.0.The suspensions for infiltration were prepared by mixing the bacteria carrying the nLuc fusion, the nLuc fusion and the silencing inhibitor strain pSoup-p19 in a 1:1:1 ratio (v/v/v) and incubated at room temperature for 3 h.The bacterial suspensions were infiltrated into young N. benthamiana leaves.At 2 days post inoculation, the Nicotiana leaves were infiltrated with luciferin solution (1 mM D-fluorescein potassium salt and 0.01% [v/v] TritonX-100) and kept in the dark for 5 min, then the luciferase activity was imaged with a NightShade LB985 Plant Imaging System (Berthold Technologies).

Co-immunoprecipitation assays
To test the protein interaction between BON1 and SERK4 and BRI1, the coding sequences of BON1, SERK4, and BRI1 were inserted into the pSuper1300-35S-HA, pSuper1300-35S-3xFlag-mCherry, and pSuper1300-35S-GFP vectors, respectively.The resulting constructs were co-transfected in Col-0 protoplasts prepared following previously described methods 74 .Transfected protoplasts were incubated for 18 h at room temperature and used for protein extraction as described above.The supernatants were incubated with anti-HA beads (Lablead, HNM-25-1000) for 4 h at 4 °C.The collected beads were washed four times with 1× Tris-buffered saline (TBS) buffer, and the coimmunoprecipitated proteins were eluted from the beads in elution buffer (0.1 M glycine-HCl, pH 3.0).

Subcellular localization assay
The coding regions of BON1, SERK3, and SERK4 were inserted into the pRPTL2-35S-eGFP vector.The resulting constructs were transformed into the Arabidopsis protoplasts prepared following previous described methods 74 .Transfected protoplasts were incubated for 18 h at room temperature.The subcellular localization of BON1 and SERK4 was analyzed in the Col-0 wild-type Arabidopsis protoplasts, and the subcellular localization of SERK3 and SERK4 were compared and analyzed in pad4-1 or bon1-1bon2-2bon3-3pad4-1 mutant protoplasts.Localization of all fusion proteins were examined under confocal microscopy (Nikon A1+).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 2 |
Fig. 2 | ZmBON1 is involved in BR signaling in maize.a Leaf angles of KN5585 and Zmbon1-1.Scale bars, 3 cm.b Weight per hundred kernels of KN5585 and Zmbon1-1 mutant.Significant differences were determined by two-tailed Student's t-tests, Asterisks indicate statistically significant differences (***P < 0.001, n = 3 biologically independent samples, ±SD).Red dots represent individual data points.c Root elongation phenotypes of KN5585 and Zmbon1-1 plants under mock and 100 nM eBL treatments.Scale bars, 5 cm.d Primary root length of KN5585 and Zmbon1-1 plants under 20 and 100 nM eBL treatments.Data are displayed as box and whisker plots with individual data points.The whiskers represent maximum and minimum values, the center line represents the median and the box limits are the 25th and 75th percentiles.Colored dots represent individual data points.n represents the number of plants.Different letters indicate significant difference between genotypes, which were determined by one-way ANOVA at P < 0.01.e Relative expression levels of ZmCPD and ZmBRD1 in KN5585 and Zmbon1-1 at 4-leaf stage.Significant differences were determined by two-tailed Student's t-tests, asterisks indicate statistically significant differences (*P < 0.05, n = 3 biologically independent samples, ±SD).Red dots represent individual data points.Source data are provided as a Source data file.

Fig. 7 |
Fig.7| Proposed model for the role of copine proteins in BR signaling.In the wild type, BONs interact with SERKs, which are spatially separated from BRI1.Upon BR perception, SERKs move close to BRI1 with the assistance of BONs to form the BRI1-SERKs complex.Then, BRI1 is fully activated by reciprocal phosphorylation of BRI1 and SERKs, and the BR signal is amplified to induce an efficient BR response.In the bon mutants, the interactions between BRI1 and SERKs were affected, resulting in attenuated reciprocal phosphorylation between BRI1 and SERKs, leading to an attenuated BR response.